The goal of this work is to describe the biochemical mechanism for the functional instability of the serine protease human tryptase and to define the relevance of this mechanism to the regulation of tryptase in vivo. Serine proteases in blood and inflammatory cells play important roles in host defense. The enzymatic activity of these proteases is tightly regulated by other proteins which act as physiological inhibitors; the absence of such regulation can have serious pathological consequences. Enzymatically active tryptase is stored in large amounts within the secretory granules of mast cells, and is released upon stimulation of these cells. However, regulation of tryptase is a mystery because no physiological inhibitor has been identified. Tryptase exhibits many features not seen in other serine protease, namely i) a tetrameric structure composed of four catalytic subunits, ii) rapid and spontaneous activity loss under physiological conditions, and iii) stabilization by interactions with heparin, a highly sulfated glycosaminoglycan also stored in mast cell granules. These features may function in an integrated manner to regulate the activity of the protease. Studies of the properties associated with activity loss suggest that it is a reversible process involving limited conformation changes important to the active site structure of all serine proteases, Physical, kinetic and spectral studies of native tryptase and newly produced recombinant/mutant tryptases are proposed to identify more completely the structural changes production the functional instability of tryptase and to establish the contribution of tetramer dissociation to activity loss. Physical studies will be utilized to establish the affinity and rate constraints for the interaction of tryptase with heparin, and mutational studies will be used to identify the heparin binding site. These studies will establish the structural basis for the intrinsic instability of tryptase and the mechanism by which heparin exerts its stabilizing effects. The rate and affinity constants for the interaction with heparin will define the functional life-time of tryptase after secretion from the mast cell. Understanding the structural properties responsible for the functional instability may provide new targets for therapeutic inhibitors.